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Abstract

Introduction

Systemic lupus erythematosus (SLE) is characterized by B cell hyper-activation and
auto-reactivity resulting in pathogenic auto-antibody generation. The phenotypic analysis
of blood B cell subsets can be used to understand these alterations.

Results

SLE patients exhibited several abnormalities in the distribution of these B cell subsets,
including elevated levels of DN memory B cells and PCs, and decreased CD27 IgD IgM
B cells. Active SLE patients also showed decreased presence of S memory B cells and
increased proportions of naive B lymphocytes. Nevertheless, when the patients in remission
who did not require treatment were studied separately, the only remaining abnormality
was a reduction of the CD27 IgD IgM B cell-subset detectable in most of these patients.
The level of reduction of CD27 IgD IgM B cells was associated with elevated values
of serum SLE auto-antibodies. Further analysis of this latter B cell-subset specifically
showed increased expression of CD80, CD86, CD95, 9G4 idiotype and functional CXCR3
and CXCR4.

Conclusions

The presence of a reduced blood CD27 IgD IgM B cell-subset, exhibiting an activated
state and enriched for auto-reactivity, is a consistent B cell abnormality in SLE.
These findings suggest that CD27 IgD IgM B lymphocytes play a role in the pathogenesis
of this disease.

Materials and methods

Patients and control populations

Heparinized blood samples (10 ml) were obtained from 31 normal healthy donors (16
women and 15 men; mean age 40 ± 2 years (range 26 to 59)) and 69 consecutive SLE patients
(63 women and 6 men; mean age 43 ± 2 years (range 19 to 77)), who fulfilled the American
College of Rheumatology criteria for SLE. Disease activity was defined by the SLE
Disease Activity Index (SLEDAI). Treatments received by the patients and SLEDAI score
at the time of analysis are shown in Table 1.

Table 1. Treatments received by the patients and SLEDAI score at the time of analysis

Patients 9, 17, 25, 29, 32, 38, 41, 43, 45, 46, 50, 53, 61, 64 and 68 had not received
any treatment during a period of, at least, three months before the analysis. The
sample from patient 29 was obtained just before she received cyclophosphamide bolus
therapy and, consequently, her results are not included in the untreated group. Patients
and healthy donors were informed of the objective of the study and gave their consent
according to the Declaration of Helsinki. Approval for this study was obtained from
the Institutional Review Board. (Comité Ético, Hospital Universitario Puerta del Mar).

Chemotaxis assays

Chemotaxis to several chemokines of CD27 IgD IgM B cells from control and SLE individuals
was determined as previously described [8]. Briefly, chemotaxis assays were carried out in 24-well plates with transwell inserts
(5-μm pore size; Costar Corning, Corning, NY, USA). RPMI 1640 medium supplemented
with BSA 0.5% was used as assay medium. PBMCs were diluted in medium at a concentration
of 10 × 106 cells/mL. The lower transwell chamber was filled with 600 μL of medium either alone
(control) or containing 1 μg/mL of CXCL12 (SDF-1α), CXCL10 (IP-10) or CCL3 (MIP-1α)
(PeproTech, London, UK), and then 100 μL of the cell suspension was added to the upper
chamber. Cells were allowed to migrate for four hours at 37°C. Finally, the cells
were collected from the lower chamber and were stained and quantified by FACS. The
total number of migrated CD19+ CD27+ IgD+ B cells was evaluated. Specific migration
was calculated as the number of cells that migrated in response to the stimuli divided
by the number of cells that migrated in response to medium (migration index).

Statistical methods

Differences between groups were analyzed with the Student's t test and Mann-Whitney test. Comparison of data included in Figure 1C was performed by ANOVA testing, followed by Tukey's post-hoc analysis for pairwise
comparisons. P-values lower than 0.05 were considered statistically significant.

Figure 1.Comparative analysis of blood B cell-subsets from SLE patients and healthy controls. CD19+ cells can be distributed into different B cell-subsets according to their
additional expression of CD27 and IgD. A. Representative CD27-IgD dot plots from a control and a SLE patient indicating the
distribution and percentages of these B cell-subsets: CD27-IgD+ (naïve), CD27+IgD-
(S memory), CD27+IgD+ (CD27 IgD IgM), and CD27-IgD- (DN memory) B lymphocytes, and
CD27highIgD- cells (PC). B. Scatter plots represent the percentages of these B cell-subsets in 31 controls (open
circles) and 69 SLE patients (closed circles). The mean of each set of values is shown
as a horizontal line. P values (Student's t test) are included. C. Scatter plots represent the percentage of each B cell-subset in controls and in
SLE patients distributed according to disease activity (12 active and 57 inactive),
and to the treatment (54 treated and 14 untreated). Symbols representing the control
and the different SLE subgroups are displayed (bottom right panel). P values were calculated for the difference between the two pairs of SLE subgroups and
between SLE subgroups and the control group. P values lower than 0.05 were considered statistically different (indicated as *).

Results

Blood B cell-subsets alterations in SLE patients

Blood B cells, including PCs, can be identified as CD19+ cells [2]. The absolute number of blood B cells (CD19+ cells) in the present group of SLE patients
was significantly lower than in normal subjects (82.0 ± 8.2 cells/μl versus 144.7
± 29 cells/μl, for SLE and healthy controls, respectively; mean ± SEM; P < 0.01). The additional staining of these cells for surface CD27 and IgD molecules,
and the subsequent FC analysis, allows the distinction of five B cell-subsets. Figure
1A shows an example of the phenotypic analysis of these blood B cell-subsets in a control
individual and in one SLE patient (FC dot plots), and Figure 1B summarizes the results obtained from all the SLE patients and the healthy controls.
As can be seen, SLE samples showed several alterations in the distribution of these
B cell-subsets, including increased proportions of DN memory B cells (CD27- IgD-)
and PC (CD27++ IgD-) and decreased CD27 IgD IgM B cells (CD27+ IgD+); this latter
finding was the most consistent. The results for blood naive B cells (CD27- IgD+)
and S memory B cells (CD27+ IgD-) were similar in SLE patients and controls.

Effect of disease activity and remission

The effect of disease activity on the described alterations of the B cell-subsets
was examined next. Accordingly, SLE patients were distributed into two groups: one
group consisted of inactive and mildly active cases (SLEDAI range 0 to 5; 0.95 ± 0.2,
mean ± SEM; N = 57); the second group consisted of moderate to highly active patients
(SLEDAI range 8 to 19; 11.17 ± 1.09, mean ± SEM; N = 12). Figure 1C shows that both groups exhibited abnormally high figures of DN memory B cells and
PC, but they differed in that the more active SLE cases additionally showed significantly
lower CD27 IgD IgM B cells and S memory B cells, and abnormally high naive B cells.

The relevance of the described B cell-subset imbalances during the course of the disease
was then analyzed. To this end, the group of SLE patients that remained in a controlled
and favorable phase of the disease, requiring no treatment at the time of the study,
was explored separately, and compared with the group of patients receiving treatment.
The clinical characteristics of the group of untreated patients are depicted in Table
2.

Table 2. Summary of clinical characteristics of the group of untreated patients

Figure 1C shows that, the elevation of DN memory B cells and PC proportions previously observed
when all the SLE cases were considered (Figure 1B), was normalized in the group of untreated patients. In contrast, most of the patients
included in the untreated group (9 out of 14) still showed a decrease in the proportion
of CD27 IgD IgM B lymphocytes (Figure 1C), although, as a whole, the difference between this group and the controls was non-significant
(Tukey's test, P = 0.09).

In an attempt to gain a deeper insight into this particular B cell-subset, a broad
phenotypic analysis was performed on them, and the results obtained in SLE patients
and healthy controls were compared. The phenotype of normal CD27 IgD IgM B lymphocytes
has been in part previously reported [2,9,10]. Figure 3A shows an example of the histogram of expression of several B cell markers on CD27
IgD IgM B cells, and Figure 3B summarizes the results obtained in all the SLE and control blood samples analyzed.
The majority of SLE and control CD27 IgD IgM B cells similarly expressed CD19, CD20,
CD21, CD22, CD35, CD49 d, CD50, CD54, CD62L and TACI (data not shown) and surface
IgM. In contrast, Figure 3A and 3B show that SLE CD27 IgD IgM B cells expressed higher levels of the co-stimulatory
molecules CD80 and CD86, the death receptor CD95, and the chemokine receptors CXCR3
and CXCR4. Figure 3C also shows that the frequency of 9G4+ cells detected in this B cell-subset was clearly
higher in SLE than in controls. This distinctive pattern of molecule expression was
equally detected in CD27 IgD IgM B cells from patients with active disease or in remission
stage (data not shown). Interestingly, the comparison of the same phenotypic study
performed on naive lymphocytes and DN and S memory B cells from healthy controls and
SLE patients revealed that, they were appreciably similar, with the exception of a
higher proportion of CD95-expressing DN memory B cells in SLE patients (Figure 4). Therefore, the observation of relevant differences in phenotype between SLE and
control blood B cells was essentially restricted to the CD27 IgD IgM B cell-subset.
Finally, the functionality of increased CXCR4 and CXCR3 expression on CD27 IgD IgM
B cells in SLE patients was examined in a chemotaxis assay. As can be seen in Figure
3D, CD27 IgD IgM B cells from healthy controls and SLE patients migrated to the CXCR4
ligand CXCL12, although these latter cells exhibited markedly higher activity (P < 0.02). In addition, cells of healthy control and SLE patients also migrated to CXCL10,
a ligand of CXCR3. Again, the chemotactic activity of SLE CD27 IgD IgM B lymphocytes
was higher, although, in this case, the observed increase was not significant. SLE
and healthy control CD27 IgD IgM B cells did not express the chemokine receptor CCR5
(data not shown) and, as expected, no chemotaxis was observed when CCL3, a ligand
of CCR5, was tested.

Figure 3.Comparative phenotypic analysis of blood CD27 IgD IgM B cell-subsets (CD27+IgD+) from
controls and SLE patients. This B cell-subset was examined for the surface expression of different molecules.
A. Representative histograms depict the expression of IgM, CD80, CD86, CXCR3, CXCR4
and CD95 for a healthy control and a SLE patient. For each baseline plot, negative
isotypic antibody controls are superimposed in dotted lines. B. Bar histograms show the percentages of positive CD27+IgD+ B cells (upper) and the
mean fluorescence intensity (lower) for each marker in healthy controls (N = 10; open
bars) and SLE patients (N = 10; grey bars). C. Expression of 9G4 idiotype in healthy controls (N = 6) and SLE patients (N = 22)
is shown. A representative histogram is shown. Results represent the mean ± SEM. P values were calculated using the Mann-Whitney test. P values lower than 0.05 were considered statistically different (marked with an asterisk).
D. A comparison is shown of the chemotaxis of CD27 IgD IgM B cells from healthy controls
and SLE patients induced by CXCL12, CXCL10 and CCL3. Migration in the absence of stimuli
is represented as a dotted line. Results are expressed as a migration index and represent
the mean ± SEM (N = 4). P-values were calculated using the Mann-Whitney test. P < 0.05 was considered statistically significant. Asterisks represent significant differences
between healthy controls and SLE patients. Hashes indicate significant differences
in chemokine-induced migration with respect to medium alone.

Discussion

The present study demonstrates that the distribution of blood B cell-subsets in SLE
patients exhibits a variety of alterations including elevated proportions of DN memory
B lymphocytes and PC, and decreased CD27 IgD IgM B lymphocytes. In addition, active
patients show decreased S memory and increased naïve B lymphocytes, indicating that
patients in more active phases exhibit more abnormalities in the distribution of these
B cell-subsets, and these abnormalities are more pronounced. These data are consistent
with previous reports [3,5-7] and, taken together, indicate that, in SLE patients, the B cell compartment undergoes
profound alterations and imbalances that are noticeable in the distribution of the
normal B cell-subsets present in the circulation. Moreover, the results observed in
the group of patients in remission (untreated patients, Table 2) show that a decreased proportion of CD27 IgD IgM B lymphocytes is the only B cell-subset
alteration persisting in the majority of these cases. Serum SLE auto-Ab are relevant
clinical parameters in this disease. Present data reveal that the level of elevation
of these auto-Ab correlates with the intensity of reduction of CD27 IgD IgM B lymphocytes
in this disease. Further analysis indicates that CD27 IgD IgM B cells from SLE patients
exhibit higher expression of CD95, CD80, CD86, CXCR3 and CXCR4. Chemotaxis assays
confirm that the increased expression of CXCR4 and CXCR3 observed in SLE CD27 IgD
IgM B cells is functional, as the migration capacity to the appropriate ligands exhibited
by these lymphocytes is higher in SLE patients' than in healthy controls. Increased
CD95 expression has been previously reported in blood DN memory B cells from SLE patients
[11]. Present results show that this observation can be also extended to CD27 IgD IgM
B cell-subset (Figures 1 and 4). In addition, increased expression of CD80 and CD86 in certain SLE circulating B
lymphocytes has been previously reported [12], although the use of a B cell marker selection profile different from that employed
in the present study makes the comparison of these results difficult. Present data
are consistent with the notion that the SLE patients' CD27 IgD IgM B cells distinctively
are in an activated state, exhibiting a potentially higher capacity to migrate and
to interact with T cells. Taken together, the present findings indicate that a decreased
proportion of blood CD27 IgD IgM B lymphocytes appears to be an alteration that is
permanent in SLE patients, irrespective of disease activity; in addition, these cells
exhibit an activated phenotype. These observations suggest that CD27 IgD IgM B cell-subset
might play a role in the patho-physiology of SLE.

Previous studies have established that the repertoire of mature naïve B lymphocytes
is enriched for self-reactive clones in SLE patients [13,14]. This observation indicates that SLE patients exhibit defective B cell tolerance
checkpoints present in normal subjects [15] and, in consequence, the emergence of an increased frequency of these auto-reactive
naive B cells is probably relevant in the pathogenesis of SLE. However, a possible
role of CD27 IgD IgM B lymphocytes in the state of auto-reactivity characteristic
of this disease has been less studied. This is, at least in part, due to the fact
that the origin, nature and functional significance of normal CD27 IgD IgM B cells
remain subject of debate [7,9,10]. These lymphocytes were defined as non-switched (IgM+ IgD+) memory B cells based
on the findings that they express the putatively memory B cell marker CD27 [2], and harbor IGV genes exhibiting a low but detectable number of somatic mutations, an event classically
restricted to post-germinal center (GC) memory B cells [2,9]. Nevertheless, the observation of normal quantities of mutated CD27 IgD IgM B lymphocytes
in immunodeficient patients that lack GC formation and conventional switched memory
B cells, indicates that the generation of the cell-subset under study can be GC-independent;
hence, the IGV gene somatic mutations present in this B cell-subset are thought to represent a pre-immune
diversification process [9,16]. Human CD27 IgD IgM B lymphocytes have been associated with the spleen marginal zone,
and they appear to be involved in the production of natural antibodies [9,16]. The mechanisms that determine the somatic mutations occurring in CD27 IgD IgM B
cells remain to be clarified, although it has been recently shown that this B cell
subset can express activation-induced cytidine deaminase (AID), either during a transient
post-natal phase [17], or upon Toll Like Receptors (TLR)-engagement [18]. Under this latter condition, CD27 IgD IgM B cells are capable of differentiating
into IgG-secreting plasma cells [18]. Interestingly, normal human CD27 IgD IgM B lymphocytes exhibit a frequency of auto-reactive
BCR much lower than that observed in naive B lymphocytes, suggesting that an additional
checkpoint for preventing self-reactivity exists at this level [19]. In this context, it is reasonable to think that a failure of this BCR control process
would necessarily lead to the emergence within this B cell-subset of auto-reactive
clones. It is well-established that the immunoglobulin (Ig) idiotype recognized by
the 9G4 mAb is enriched in auto-reactive B cell populations, including anti-DNA Ab-bearing
B cells occurring in SLE patients [20]. Accordingly, the presence of B lymphocytes containing the 9G4 idiotype in their
surface Ig was examined in the blood CD27 IgD IgM cell-subset. Present data reveal
that the frequency of 9G4+ cells detected in this B cell-subset was, on average, five
times higher in SLE than in controls. This result indicates that SLE patients show
a defective control on the appearance of auto-reactive clones within the circulating
CD27 IgD IgM B cell-subset. The cause of this failure remains unknown. It is conceivable
that these auto-reactive cells, after appropriate self-antigen recognition, maybe
in combination with TLR-activation [18], would undergo activation and migration toward lymphoid tissue where they could progress
into further differentiation, giving rise to auto-reactive switched memory B cells
and PCs. This explanation is consistent with the findings reported here. Thus, a permanent
decrease of circulating CD27 IgD IgM B cells could be the result of self-antigen activation
of auto-reactive B cells contained in elevated proportions in this subset, and their
subsequent recruitment into lymphoid tissues. In fact, 9G4+ activated B cells and
PCs have previously been detected in lymphoid tissues (spleen, tonsil GC and bone
marrow) from SLE patients, but not from normal subjects [21]. In conclusion, CD27 IgD IgM B lymphocytes might play a role in the complex B cell
alteration causing SLE.

Conclusions

This study shows that the presence of a reduced blood CD27 IgD IgM B cell-subset,
exhibiting an activated state, an increased capability to migrate towards CXCR4 ligand
and enriched for auto-reactivity is a prominent B cell abnormality in SLE. In addition,
higher levels of ANA, anti-dsDNA and anti-ENA Ab were associated with lower numbers
of CD27 IgD IgM B cells in SLE patients. These findings suggest that CD27 IgD IgM
B lymphocytes play a role in the pathogenesis of this disease.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

BRB carried out the acquisition of data, analysis and interpretation of data and statistical
analysis. ARA participated in the acquisition of data. JJPV participated in the acquisition
of data and in patients' collection. CR participated in analysis and interpretation
of data and manuscript preparation. JAB performed the study design and the manuscript
preparation. All authors read and approved the final manuscript.

Acknowledgements

Special thanks to Professor F Stevenson for the generous gift of 9G4 rat mAb.

This work was supported by the Fondo de Inverstigaciones Sanitarias of Spain (PI05/2406
and PI08/1618) and by the Junta de Andalucía of Spain (CTS02840).